Cyclic peptide antifungal agents and process for preparation thereof

ABSTRACT

Provided are compounds of the formula (1):  
                 
 
wherein R′ is hydrogen, methyl or NH 2 C(O)CH 2 —; 
R″ and R′″ are independently methyl or hydrogen; 
         R adn R y  are independently hydroxy or hydrogen;    R 1  is hydroxy, hydrogen, or hydroxysulfonyloxy;    R 7  is hydroxy, hydrogen, hydroxysulfonyloxy or phosphonooxy;    R 2  is a novel acyl side chain. Also provided are novel formulations, methods of inhibiting fungal and parasitic activity, and a process for preparing dideoxy (R═H) forms of the compounds.

This application is a continuation-in-part of U.S. application Ser. No.07/992,390 filed Dec. 16, 1992, which is a continuation-in-part of U.S.application Ser. No. 07/854,117 filed Mar. 19, 1992

BACKGROUND OF THE INVENTION

This invention relates to cyclic peptide antifungal agents. Inparticular, it relates to acyl derivatives of the echinocandin class ofcyclic peptide antifungal agents; to methods for treating antifungal andparasitic infections, and to formulations useful in the methods.

The compounds provided by this invention are semi-synthetic antifungalagents in that they are derived from the cyclic peptide antifungalswhich are produced by culturing various microorganisms. A number ofcyclic peptide antifungals are known. Among these are echinocandin B(A30912A), aculeacin, mulundocandin, sporiofungin, L-671,329, FR901379,and S31794/F1. All such antifungals are structurally characterized by acyclic hexapeptide core, or nucleus, the amino group of one of thecyclic amino acids bearing a fatty acid acyl group forming a side chainoff the core or nucleus. For example, echinocandin B has a linoleoylside chain while aculeacin has a palmitoyl side chain. These fatty acidside chains of the cyclic hexa-peptides can be removed by enzymaticdeacylation to provide the free nucleus. (Formula (1), hereinafter,wherein R₂ is hydrogen.) Reacylation of the amino group of the nucleusprovides semisynthetic. antifungal compounds. For example, theechinocandin B nucleus provides a number of antifungal agents whenreacylated with certain unnatural side chain moieties (see Debono, U.S.Pat. No. 4,293,489). Among such antifungal compounds is cilofungin whichis represented by the formula (1) wherein R is methyl, R₁ is hydrogenand R₂ is p-(n-octyloxy)benzoyl.

Enzymatic deacylation of the cyclic hexapeptides is carried out withdeacylase produced by the organism Actinoplanes utahensis and relatedmicroorganisms as described by Abbott et al., U.S. Pat. No. 4,293,482.

The present invention provides acylated cyclic hexapeptides havingunique side chain acyl groups which, inter alia impart enhancedantifungal and antiparasitic potency e.g. against pathogenic strains ofCandida albicans. Also provided is a process for removing the aminal andbenzylic hydroxy groups to result in a dideoxy compound of formula (1)(R═H).

SUMMARY OF THE INVENTION

The compounds provided by this invention are represented by thefollowing formula (1):

wherein

-   -   R′ is hydrogen, methyl or NH₂C(O)CH₂—;    -   R″ and R′″ are independently methyl or hydrogen;    -   R and R^(y) are independently hydroxy or hydrogen;    -   R₁ is hydroxy, hydrogen, or hydroxysulfonyloxy;    -   R₇ is hydroxy, hydrogen, hydroxysulfonyloxy or phosphonooxy; and        I) R₂ is a substituted benzoyl group represented by the formula        wherein    -   A) R₃ is a polyoxa-alkyl group represented by the formula        —O—(CH₂)_(m)—[O—(CH₂)_(n)]_(p)—O—(C₁-C₁₂ alkyl)        wherein m and n are integers of from 2 to 4, and p is 0 or 1; or    -   B) R₃ is an unsaturated hydrocarbon group represented by the        formula        —Y—(C₁-C₁₂ alkyl)        wherein Y is —C≡C— or —CH═C—; or    -   C) R₃ is a group of the formula —O—(CH₂)_(m)-G wherein m is as        defined and G is C₇-C₁₀ bicycloalkyl or C₇-C₁₄ tricycloalkyl; or    -   D) R₃ is quinolyl; or        II) R₂ is an acyl group represented by the formula        wherein    -   Z is —O—, —C≡C—, —CH═CH—, —CH₂—CH₂—, —CH₂—, or a carbon to        carbon bond;    -   A) R₄ is hydrogen, C₂-C₁₂ alkynyl, C₂-C₁₂ substituted alkynyl,        C₃-C₁₂ cycloalkyl, C₇-C₁₀ bicycloalkyl, C₇-C₁₄ tricycloalkyl,        C₁-C₁₂ alkoxy, C₃-C₁₂ cycloalkoxy, naphthyl, pyridyl, thienyl,        benzothienyl, quinolyl or phenyl; or    -   B) R₄ is phenyl substituted by amino, C₁-C₁₂ alkylthio, halogen,        C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂ substituted        alkyl, C₂-C₁₂ substituted alkenyl, C₂-C₁₂ substituted alkynyl,        C₁-C₁₂ alkoxy, trifluoromethyl, phenyl, substituted phenyl,        phenyl substituted with a polyoxa-alkyl group represented by the        formula        —O—(CH₂)_(m)—[O—(CH₂)_(n)]_(p)—O—(C₁-C₁₂ alkyl)        wherein m, n and p are as defined; or    -   C) R₄ is phenyl substituted with C₁-C₆ alkoxy substituted by        fluoro, bromo, chloro or iodo; or    -   D) R₄ is C₁-C₁₂ alkoxy substituted with C₃-C₁₂ cycloalkyl,        C₇-C₁₀ bicycloalkyl, C₇-C₁₄ tricycloalkyl, C₂-C₁₂ alkynyl,        amino, C₁-C₄ alkylamino, di-(C₁-C₄ alkyl)amino, C₁-C₁₂        alkanoylamino, phenyl substituted with a polyoxa-alkyl group        represented by the formula        —O—(CH₂)_(m)—[O—(CH₂)_(n)]_(p)—O—(C₁-C₁₂ alkyl)        wherein m, n and p are as defined; or    -   E) R₄ is C₁-C₁₂ alkoxy substituted with a group of the formula        wherein R₈ is C₁-C₆ alkoxy optionally substituted with phenyl;        or    -   F) R₄ is a group represented by the formula        —O—(CH₂)_(p′)—W—R₅        wherein p′ is an integer of from 2 to 4; W is pyrrolidino,        piperidino or piperazino, and R₅ is hydrogen, C₁-C₁₂ alkyl,        C₃-C₁₂ cycloalkyl, benzyl or C₃-C₁₂ cycloalkylmethyl; or    -   G) R₄ is a group represented by the formula        —Y—R₆        wherein Y has the same meanings defined above; and    -   R₆ is C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl; C₃-C₁₂ cycloalkyl,        C₇-C₁₀ bicycloalkyl, C₇-C₁₄ tricycloalkyl, phenyl, C₃-C₁₂        cycloalkenyl, naphthyl, benzothiazolyl, thienyl, indanyl,        fluorenyl, phenyl substituted by amino, C₁-C₁₂ alkylthio,        halogen, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂        alkoxy, trifluoromethyl, —O—(CH₂)p′-W—R₅, or C₁-C₆ alkoxy        substituted by fluoro, bromo, iodo or chloro; or    -   R₆ is a phenyl substituted by a polyoxa-alkyl group represented        by the formula        —O—(CH₂)_(m)—[O—(CH₂)_(n)]_(p)—O—(C₁-C₁₂ alkyl)        wherein m, n and p are as defined above; or        III) R₂ is a group having the formula        wherein R^(x) is C₁-C₁₂ alkoxy or a polyoxa-alkyl group        represented by the formula        —O—(CH₂)_(m)—[O—(CH₂)_(n)]_(p)—O—(C₁-C₁₂ alkyl)        wherein m, n and p are as defined above; or        IV) R₂ is a group having the formula        wherein R₉ is phenyl, C₁-C₁₂ alkyl, or C₁-C₁₂ alkoxy; or        V) R₂ is naphthoyl substituted with R₄; and the pharmaceutically        acceptable non-toxic salts thereof;    -   with the proviso that when        -   R′ is methyl or NH₂C(O)CH₂—;        -   R″ is methyl;        -   R′″ is methyl;        -   R^(y) is hydroxy;        -   R is hydroxy; and either a) or b):    -   a) R₁ is hydroxysulfonyloxy and R₇ is hydroxy,        hydroxysulfonyloxy or phosphonooxy;    -   b) R₁ is hydrogen or hydroxysulfonyloxy and R₇ is        hydroxysulfonyloxy or phosphonooxy;        -   R₂ is not            wherein R₃ is            —O—(CH₂)_(m)—[O—(CH₂)_(n)]_(p)—O—(C₁-C₁₂ alkyl)            wherein p=0; nor            wherein Z is a carbon to carbon bond or —O— and R₄ is C₁-C₁₂            alkoxy; nor            -   iii) naphthoyl substituted by R₄ wherein R₄ is hydrogen,                phenyl, or C₁-C₁₂ alkoxy.                Also provided are formulations and methods for                inhibiting parasitic and fungal activity which employ                the compounds of the invention, and a process for                preparing the dideoxy form of the compounds.

DETAILED DESCRIPTION

The term: “C₁-C₁₂ alkyl” refers to the straight or branched chain alkylhydrocarbon groups such as, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl,nonyl, decyl, undecyl and dodecyl groups; and the like.

The term “C₂-C₁₂ alkenyl” refers to groups such as vinyl,1-propene-2-yl, 1-butene-4-yl, 1-pentene-5-yl, 1-butene-1-yl, and thelike.

The term “C₂-C₁₂ alkynyl” refers to such groups as ethynyl, propynyl,pentynyl, butynyl and the like.

The term “C₁-C₁₂ alkylthio” refers to such groups as methylthio,ethylthio, t-butylthio, and the like.

The term “C₁-C₁₂ alkoxy” refers to the straight or branched chainoxyalkyl groups such as, e.g. methoxy, ethoxy, propoxy, butoxy, heptoxy,octyloxy, dodecyloxy, and the like.

The term C₃-C₁₂ cycloalkoxy” refers to such groups as cyclopropoxy,cyclobutoxy and the like.

The term “C₃-C₁₂ cycloalkenyl” refers to such groups as cyclopropenyl,cyclobutenyl, cyclopentenyl, and the

The term “C₁-C₁₂ substituted alkyl,” “C₂-C₁₂ substituted alkenyl”, and“C₂-C₁₂ substituted alkynyl”, denotes the above substituted one or twotimes with halogen, hydroxy, protected hydroxy, amino, protected aminoC₁-C₇ acyloxy, nitro, carboxy, protected carboxy, carbamoyl,carbamoyloxy, cyano, methylsulfonylamino, phenyl, substituted phenyl, orC₁-C₁₂ alkoxy.

The term “substituted phenyl” is represented by a phenyl groupsubstituted with one, two, or three moieties chosen from halogen,hydroxy, protected hydroxy, cyano, nitro, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy,carboxy, protected carboxy, carboxymethyl, hydroxymethoyl, amino,aminomethyl trifluoromethyl or N-(methylsulfonylamino)

The term “C₃-C₁₂ cycloalkyl” refers to such groups as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The term “C₁-C₄ alkylamino” refers to such groups as methylamino,ethylamino, n-butylamino and the like.

The term “di-(C₁-C₄ alkyl)amino” refers to such groups as dimethylamino,diethylamino, di-n-propylamino, di-n-butylamino, methylethylamino,methyl-n-butylamino, and like tertiary amino groups.

The term “C₁-C₁₂ alkanoylamino” refers to such groups as acylaminogroups derived from the C₁-C₁₂ carboxylic acids and are exemplified byformamido, acetylamino, propionylamino, butyrylamino, and the like.

The term “C₃-C₁₂ cycloalkylmethyl” refers to those C₃-C₇ cycloalkylsdescribed above further substituted by methyl.

The terms “C₇-C₁₀ bicycloalkyl” and “C₇-C₁₄ tricycloalkyl” refer to suchgroups as bicyclo[2.2.1.]hept-2-yl, bicyo[2.2.1.]hep-4-en-2-yl,bicyclo[3.3.1.]nona-3-yl, bicyclo[3.3.1.]nona-2-yl,bicyclo[3.2.1.]oct-2-yl, bicyclo[2.2.2.]oct-2-yl,bicyclo[2.2.2]oct-5-en-2-yl, adamantyl and the like.

The term “dideoxy” refers to compounds of the formula (1) wherein R═H.

The term “inhibiting”, such as used in relation to the methods forinhibiting parasitic and fungal activity, is defined to mean its normaldefinition, i.e., to stop, retard or prophylactically hinder or prevent.

The term “activity”, as used in relation to parasitic and fungalactivity, includes growth thereof and attending characteristics andresults from the existence of the parasite or fungus.

The term “contacting”, as used in relation to the methods for inhibitingparasitic and fungal activity by contacting a compound of the inventionwith a parasite or fungus, is defined to mean its normal definition.However, the term does not imply any further limitations to the process,such as by mechanism of inhibition, and the methods are defined toencompass the spirit of the invention, which is to inhibit parasitic andfungal activity by the action of the compounds and their inherentanti-parasitic and anti-fungal properties, or in other words, thecompounds, used in the method are the causative agent for suchinhibition.

Examples of acyl groups represented by R₂ in formula (1) are benzoylsubstituted by polyoxa-alkyl groups such as, e.g., 2-methoxyethoxy (p=0,m=1), 2-ethoxyethoxy, 2-(2-ethoxyethoxy)ethoxy (m=2, p=1, n=2),3-(2-ethoxyethoxy)-propoxy, 3-(2-methoxyethoxy)butoxy, and like groups.

Examples of R₃ groups wherein R₂ is benzoyl substituted by anunsaturated hydrocarbon groups —Y—(C₁-C₁₂-alkyl) include e.g.,acetylenic groups —C≡C—(C₁-C₁₂ alkyl) and —CH₂═CH₂—(C₁-C₁₂ alkyl) whichmay be cis- or trans- e.g. propenyl, butenyl, hexenyl, decenyl, and thelike; propynyl, butynyl, hexynyl, undecynyl, and like alkynes.

Examples of acyl groups wherein R₂ is a group represented by the formula

are diphenyl ethers (Z=—O—), diphenyl acetylenes (Z=—C≡C—), stilbenes(Z=—CH═CH—), and biphenyls (Z=a carbon to carbon bond). Among examplesof such biphenyl groups, wherein Z is a carbon to carbon bond i.e. aphenyl to phenyl bond, are 4-[4-(butyloxy)phenyl]benzoyl,4-[4-(cyclobutylmethoxy)-phenyl]benzoyl,4-[4-cyclopentyl-methoxy)phenyl]benzoyl,4-[4-(cyclohexylethoxy)-phenyl]benzoyl,4-[4-(n-hexyloxy)-phenyl]benzoyl, 4-phenylbenzoyl,4-[4-(11-amino-undecyloxy)-phenyl]benzoyl,4-[4-(11-formamidoundecyloxy)phenyl]benzoyl,4-[4-(iso-pentyloxy)phenyl]benzoyl, and the like. Examples of suchdiphenyl ether acyl groups R₂ of the formula above wherein Z is anoxygen atom are 4-(4-butyloxyphenoxy)benzoyl,4-(4-hexyloxyphenoxy)benzoyl, 4-(4-ethoxyphenoxy)benzoyl,4-(4-benzyloxyphenoxy)benzoyl, 4-[4-(3-chlorobutyloxy)phenoxy]-benzoyl,4-(4-dodecyloxyphenoxy)benzoyl,4-[4-(3-di-methylaminopropoxy)phenoxy]benzoyl and the like. Examples ofdiphenylacetylene and stilbene acyl groups, R₂, wherein Z is anacetylenic bond or an ethylene bond are 4-styrylbenzoyl,4-(4-methoxystyryl)benzoyl, 4-(4-butyloxystyryl)benzoyl,4-(phenylethynyl)benzoyl, 4-(4-ethoxyphenylethynyl)benzoyl,4-(4-cyclohexyloxyphenyl-ethynyl)benzoyl, and the like. Examples of R₂acyl groups represented by the foregoing formula wherein Z is a carbonto carbon bond and R₄ is represented by the formula —O—(CH₂)_(p′)—W—R₅are 4-[4-[2-(N-cyclohexylpiperidine-4-yl)ethoxy]phenyl]benzoyl,4-[4-[2-(N-hexylpiperidine-4-yl)ethoxy]phenyl]benzoyl,4-[4-[2-(4-benzylpiperidino)-ethoxy]phenyl]benzoyl,4-[4-[2-(4-cyclohexylpiperidino)-ethoxy]phenyl]benzoyl and like diphenylacyl groups. Examples of such acyl groups wherein R₄ is represented bythe formula —Y—R₆ include 4-[4-(phenylethynyl)phenyl]benzoyl,4-[4-(phenylethynyl)-phenoxy]benzoyl, 4-[4-(hexynyl)phenyl]benzoyl,4-[4-(styryl)phenoxy]benzoyl,4-[4-(4-benzylphenylethynyl)-phenyl]benzoyl,4-[4-[4-4-methylpiperidino)ethoxy]phenyl-ethynyl]phenyl]benzoyl and likeacyl groups. Such acyl groups wherein R₄ is represented by the formula—O—(CH₂)_(p′)—W—R₅ form salts of the basic amino groups of thepiperidine and piperazine heterocyclic groups with both organic andinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid and phosphoric acid and with organic acids such as the sulfonicacids, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid,acetic acid, chloroacetic acid, trifluoroacetic acid, benzoic acid,isophthalic acid, salicylic acid, citric acid, malic acid, succinicacid, malonic acid and like acids.

The following tables contain further examples of the cyclic peptidesrepresented by the formula (1). Table 1 contains examples of cyclicpeptides wherein the acyl group R₂ is of the formula TABLE 1

R₂

The following Table 2 illustrates the compound of the formula (1)wherein R₂ is represented by the formula TABLE 2

R₂

The following Table 3 illustrates compounds of formula 1 wherein R₂ isof the formula as indicated from Table 2 and R₄ is represented by theformula —O—(CH₂)_(p)—W—R₅. TABLE 3 R₂

The acyl cyclohexapeptides represented by formula (1) exhibitantiparasitic activity, for example, they are especially active againstthe infectious fungi Candida albicans and Candida parapsilosis. Theyalso exhibit significant activity against Aspergillus fumigatus. Theyare active both in vitro and in vivo and accordingly are useful incombating systemic fungal infections.

The compounds of the invention also inhibit the growth of certainorganisms primarily responsible for opportunistic infections inimmunosuppressed individuals. For example the compounds of the inventioninhibit the growth of Pneumocystis carinii the causative organism ofpneumocystis pneumonia in AIDS sufferers.

The antifungal activity of the compounds of the invention is determinedin vitro in standard agar dilution tests and disc-diffusion testswherein minimum inhibitory concentrations of the test compoundsobtained. Standard in vivo tests in mice are used to determine theeffective dose of the test compounds in controlling systemic fungalinfections.

Tables 4A-E below contain the minimum inhibitory concentrations (MIC) inmicrograms per milliliter (mcg/ml) for compounds of the inventionagainst Candida albicans and Candida parapsilosis, and for certaincompounds, the effective dose, ED₅₀, in mice.

In Tables 4A-E, R′═CH₃, R═CH₃, R″═CH₃, R′″═CH₃, R^(y)═OH, R₇═OH andR₁═H, In Tables 4A-D, R═OH, while in Table E, R═H.

In the Table 4A, R₂ is of the formula with R₃ being as indicated in theTable 4.

In Table 4B, R₂ is of the formula

where Z is —O— and R₄ is as indicated.

Table 4C is as Table 4B, except Z is a carbon-carbon bond.

Table 4D indicates compound activities in which R₂ is as defined.

In Table 4E, dideoxy (where R=H) compounds are illustrated with R₂ asindicated. TABLE 4A MIC (mcg/ml) ED₅₀ R₃ C. alb. C. parap. (mg/kg)—O(CH₂)₂—O—(CH₂)₂—O—C₂H₅ >20 40 — —O—(CH₂)₂—O—C₅H₁₁ >20 40 ——O—(CH₂)₂—OC₇H₁₅ 10 40 30.3 —O—(CH₂)₂—O—C₈H₁₇ 2.5 80 4.4—O—(CH₂)₂—O—C₁₀H₂₁ 0.625 5 9.5 —C≡C—C₅H₁₁ 2.5 29 10.5 —CH═CH—C₆H₁₃(trans) 0.312 20 4.4 —C≡C—C₈H₁₇ 0.156 10 —

TABLE 4B MIC (mcg/ml) ED₅₀ R₄ C. alb. C. parap. (mg/kg) —O—C₄H₉ >20 40 ——O—C₆H₁₃ 1.25 >20 22.9

TABLE 4C MIC ED₅₀ (mcg/ml) (mg/ C. R₄ C. alb. parap. ml) —O—C₄H₉ 0.78 100.84 —O—CH₂—cyclobutyl 0.312 10 2.50 —O—CH₂—cyc1opentyl 0.039 2.5 1.20—O—C₅H₁₁ 0.156 0.625 1.86 —O—C₆H₁₃ 0.039 1.25 1.10 —O—CH₂CH₂—cyclohexyl0.039 20 1.6 —O—CH₂—CH(C₂H₅)—C₂H₅ 0.039 2.5 4.6 —O—CH₂—CH₂—CH(CH₃)₂0.309 5 2.00 —O—CH₂—CH₂—C(CH₃)₃ 0.039 2.5 2.21 —O—(CH₂)₂—O—C₅H₁₁ 1.25 200.60 —C≡C—C₄H₉ 0.039 2.5 1.20 —C≡C—C₆H₅ 0.039 0.625 0.60 —C₆H₅ 0.078 101.3 —O—(CH₂)₂—N(CH₃)₂ >20 >20 —

>20 >20 —

5 >20 3.0

0.312 40 0.64

0.039 5 0.24

TABLE 4D MIC (mcg/ml) R₂ C. alb. C. parap.

40 >80

1.25 80

0.0039 2.5

5 >80

80 >80

80 >80

10 >80

>80 >80

20 >80

10 >80

20 >80

20 >80

0.039 5

0.078 0.312

0.5 80

0.005 0.156

0.039 0.156

0.156 20

0.005 0.312

0.312 5

0.312 >80

0.078 >20

TABLE 4E MIC (mcg/ml) R₂ C. alb. C. parap.

0.039 5.0

>20 1.25

0.039 2.5

>80 >80

1.25 40

0.005 2.5

0.0098 0.625

80 >80

20 >80

40 >80

1.25 >80

>80 >80

10 >80

10 >80

5.0 >80

1.25 >80

0.078 1.25

0.039 0.125

0.156 0.625

0.156 5.0

0.625 80

0.005 0.156

0.039 0.156

The non-dideoxy compounds of the invention (formula (1) are preparedwith the amino nuclei of the cyclic hexapeptides which are representedby the formula when R₂ is hydrogen. These amino nuclei are obtained fromthe known natural products by the known enzymatic deacylation by whichthe fatty acid side chains of the natural compounds are removed. Forexample, echinocandin B which can be represented by the formula (1)wherein R′═R″═R′″=methyl, R is OH, R^(y) is hydroxy, R₁ is H, R₇ is OH,and R₂ is linoleoyl, is deacylated to provide the echinocandin B nucleus(R₂═H) with the deacylase produced by the organism Actinoplanesutahensis as described by U.S. Pat. Nos. 4,293,482 and 4,304,716.

The known natural cyclic hexapeptides which are N-deacylated to providethe amino nuclei starting materials include echinocandin B (also knownas A-30912A), aculeacin (palmitoyl side chain), tetrahydoechinocandin B(stearoyl side chain), mulundocandin (branched C₁₅ side chain),L-671,329 (C₁₆ branched side chain), S 31794/F1 (tetradecanoyl sidechain), sporiofungin (C₁₅ branched side chain) and FR901379 (palmitoylside chain). The amino nuclei obtained by the N-deacylation are thenacylated by employing known amino acylation procedures to provide theN-acyl cyclic hexapeptides represented by the formula (1) wherein R₂represents the acyl groups defined hereinabove. The acylating moiety ispreferably an active ester of the carboxylic acid RCOOH such as the2,4,5-trichlorophenyl ester. The R₂COOH precursor acids are prepared bythe hydrolysis of the nitrile R₂CN or the ester R₂COOC₁-C₄ alk. Thesenitrile and ester intermediates are prepared by known methods.

The alkoxy aromatic (ie. phenyl and biphenyl) compounds of Tables 5-10are prepared by one of the two following procedures

-   -   A. The hydroxyaromatic compound (1 equivalent) is dissolved in        acetonitrile (200-300 ml) and a base, such as potassium        t-butoxide or potassium carbonate, (1-equivalent), is added. An        alkyl bromide, iodide, or p-toluenesulfonate (1 equivalent) is        then added and the solution is refluxed for 6 hours. The solvent        is evaporated in vacu and the residue is dissolved in ether and        2N sodium hydroxide. The ether layer is dried over magnesium        sulfate and evaporated to give the alkoxyaromatic product.

B. The hydroxyaromatic compound (1 equivalent), alkyl alcohol (1equivalent), and triphenylphosphine (1 equivalent) are dissolved intetrahydrofuran (200-300 ml) and diethylazodicarboxylate (1 equivalent)is added dropwise over 10 minutes at room temperature. After 17 hoursthe solvent is removed a vacuo and the residue is dissolved in ether.This organic layer is extracted with 2N sodium hydroxide solution, driedover magnesium sulfate, and evaporated to give a product which iscrystallized from ether/pentane or, if the product contains a tertiaryamine, the hydrochloride salt is formed and crystallized frommethanol/ethyl acetate. TABLE 5

Alkyl halide or tosylate Wt. g Method R₁ R₂ Wt. g I(CH₂)₃CH₃ 9.4 A—(CH₂)₃CH₃ CN 3.2

12.3 A

CN 5.3 Br(CH₂)₂CH(CH₃)₂ 7.7 A —(CH₂)₂CH(CH₃)₂ CN 9.2

7.6 A (CH₂)₂O(CH₂)₄CH₃ CN 4.8 Br(CH₂)₄CH₃ 15.3 A —(CH₂)₄CH₃ CN 20.3

13.0 A

CN 12.2

13.1 A (CH₂)₂C(CH₃)₃ CN 11.8 BrCH₂CH(CH₂CH₃)₂ 8.5 A —CH₂CH(CH₂CH₃)₂ CN3.0 I(CH₂)₅CH₃ 10.8 A —(CH₂)₅CH₃ CN 11.4

4.2 A

CO₂CH₃ 4.5

TABLE 6

Alcohol wt. g Method R wt. g

3.6 B

6.2

6.1 B

4.3

0.5 B

0.8

0.5 B

0.5

2.3 B

1.3

9.3 B

9.6

TABLE 7

Tosylate or alcohol wt. g Method R wt. g

23.4 A —(CH₂)₂O(CH₂)₆CH₃ 20.9

25.8 A —(CH₂)₂O(CH₂)₇CH₃ 7.9

27.1 A —(CH₂)₂O(CH₂)₉CH₃ 21.0

10.0 B

13.6

TABLE 8 Alkyl halide wt. g Method

wt. g I(CH₂)₃CH₃ 6.1 A —(CH₂)₃CH₃ 12.3 I(CH₂)₅CH₃ 4.3 A —(CH₂)₅CH₃  4.7

TABLE 9

Alkylhalide or tosylate Wt. g Method R Wt. g I(CH₂)₂CH₃ 2.6 A —(CH₂)₂CH₃4.4

2.7 A —(CH₂)₂O(CH₂)₃CH₃ 2.6

2.7 A —(CH₂)₂OC(CH₃)₃ 2.6

TABLE 10

Alkylhalide or tosylate Wt. g Method R Wt. g I(CH₂)₂CH₃ 3.8 A —(CH₂)₂CH₃1.4

3.6 A —(CH₂)₂O(CH₂)₃CH₃ 5.1

4.9 A —(CH₂)₂OC(CH₃)₃ 5.2

The alkynyl and alkenyl aromatic compounds contained in Tables 11-14 areprepared by the following procedure:

An aromatic bromide, iodide, or trifluoromethane-sulfonate (1equivalent) is dissolved in acetonitrile (600 ml/0.1 mole of aromaticreactant) under a nitrogen atmosphere. An alkyne or alkene (1equivalent), triethylamine (2 equivalents), palladium dichloride (0.05equivalents), triphenylphosphine (0.1 equivalents), and cuprous iodide(0.025 equivalents) are added and the solution is refluxed for 17 hours.The solvent is removed in vacuo and the residue is slurried in ether(300 ml). Solids are removed by filtration and the filtrate is washedwith 1N hydrochloric acid solution. The organic layer is dried overmagnesium sulfate and evaporated to yield the product. TABLE 11

Acetylene or olefin wt. g wt. g R wt. g H—≡—(CH₂)₅CH₃ 12.1 28.8—C≡—(CH₂)₅CH₃ 26.2 H—≡—(CH₂)₅CH₃ 6.1 14.4 —CH═—(CH₂)₅CH₃ (trans) 0.6H—≡—(CH₂)₇CH₃ 15.2 28.8 —C≡—(CH₂)₇CH₃ 28.1

1.9 5.1

1.9 H—≡—Si(CH₃)₃ 4.3 11.5 —≡—Si(CH₃)₃ 11.2

TABLE 12

Acetylene wt. g wt. g R wt. g

1.8 6.0

2.6 H—≡—(CH₂)₃CH₃ 1.4 6.0 —C≡—(CH₂)₅CH₃ 5.1 H—≡—Si(CH₃)₃ 10.9 40.0—≡—Si(CH₃)₃ 23.3

TABLE 13

Acetylene wt. g wt. g R wt. g H—≡—(CH₂)₇CH₃ 7.6 11.3 —C≡—(CH₂)₇CH₃ 11.4

TABLE 14 wt. g Acetylene

10.5

22.2

1.2 Halide

9.7

34.4

1.2 Product

10.2

19.4

1.5

The aromatic boronic acids listed in Table 15 were prepared by thefollowing procedure:

An aromatic halide (1 equivalent) is cooled to −78° C. intetrahydrofuran solvent. Butyl lithium (1.2 equivalents) is added. After15 min triisopropyl borate (2 equivalents) is added and after 10 min ofstirring the cooling bath is removed. When the reaction has warmed toroom temperature water is added to quench the reaction followed by 1NHCl. The organic layer is removed under reduced pressure leaving a solidprecipitate which is collected by filtration. This solid is washed withhexane leaving the pure boronic acid.

The terphenyl esters listed in Table 16 were made in the followingmanner:

An aromatic boronic acid (1 equivalent), methyl 4-iodobenzoate (1equivalent), and potassium carbonate (1.5 equivalents) were mixed in anitrogen-purged toluene solution. Alternatively, the trichloro phenylester of iodobenzoate my be used. Addedtetrakis(triphenylphosphine)palladium (0.03 equivalents) and refluxedfor 7 hrs. The solution was decanted to remove the potassium carbonateand reduced in vacuo. The residue was triturated with acetonitrile andthe product solid was collected by filtration. TABLE 15 R = Br R = (OH)₂Wt. g Wt. g

10.6 6.1

31.0 12.0

10.9 4.1

13.6 5.7

5.0 1.9

TABLE 16

R Wt. (g) Wt. (g) Wt. (g) —O(CH₂)₃CH₃ 5.0 3.2 4.2 —O(CH₂)₄CH₃ 6.0 3.75.2 —O(CH₂)₅CH₃ 3.4 2.8 3.5 —O(CH₂)₂O(CH₂)₃CH₃ 3.7 3.6 3.7—O(CH₂)₂OC(CH₃)₃ 1.8 1.5 2.2

The aromatic nitrites or carboxylate esters described in Tables 5-16 canbe converted to carboxylic acids by one of the two following hydrolysisprocedures:

-   -   A. An aromatic nitrile is dissolved in ethanol and an excess of        50% sodium hydroxide solution and refluxed for 2 hours. Water is        added until a solid precipitates. The precipitate is collected        by filtration, added to dioxane and 6N hydrochloric acid        solution and refluxed for 17 hours. water is added and the        carboxylic acid product crystallizes and is collected by        filtration and dried under vacuum.    -   B. A carboxylate methyl ester is dissolved in methanol, excess        2N sodium hydroxide solution is added and the solution is        refluxed for 5 hours. The solution is made acidic with excess        hydrochloric acid and water is added until a precipitate forms.        The carboxylic acid is collected by filtration and dried under        vacuum.

The carboxylic acids are converted to 2,4,5-trichlorophenyl esters shownin Tables 17-25 by the following general procedure:

The aromatic acid (1 equivalent), 2,4,5-trichlorophenol (1 equivalent),and N,N′-dicyclohexyl-carbodiimide (1 equivalent) are dissolved inmethylene chloride. The mixture is stirred for 17 hours after which itis filtered. The filtrate is evaporated to dryness and the residue isdissolved in ether, filtered, and pentane is added until crystallizationbegins. The crystalline product is collected by filtration and driedunder vacuum. TABLE 17

2.4.5-trichlorophenol ester R wt. g wt. g —(CH₂)₃CH₃ 1.9 1.8

4.2 4.4 —(CH₂)₂CH(CH₃)₂ 3.0 1.7 —(CH₂)₂O(CH₂)₄CH₃ 2.2 13 —(CH₂)₄CH₃ 5.75.1

4.4 3.1 —(CH₂)₂C(CH₃)₃ 2.3 2.6 —CH₂CH(CH₂CH₃)₂ 1.5 0.8 —(CH₂)₃CH₃ 5.34.8

3.1 1.0

3.3 1.5

3.0 2.3

1.0 1.0

2.0 0.8

7.2 0.8

7.5 7.3

TABLE 18

2.4.5-trichlorophenol ester R wt. g wt. g

2.0 0.6 —C≡—(CH₂)₃CH₃ 1.1 0.6

TABLE 19

2.4.5-trichlorophenol R wt. g ester wt. g —(CH₂)₂O(CH₂)₆CH₃ 5.6 2.9—(CH₂)₂O(CH₂)₇CH₃ 7.8 6.6 —(CH₂)₂O(CH₂)₉CH₃ 6.4 1.3

4.0 3.2

TABLE 20

2.4.5-trichlorophenol ester R wt. g wt. g —C≡—(CH₂)₅CH₃ 4.6 3.5—CH═—(CH₂)₅CH₃ (trans) 1.2 0.5 —C≡—(CH₂)₇CH₃ 11.1 13.2

1.5 1.5

TABLE 21

2.4.5- trichlorophenol R wt. g ester wt. g —(CH₂)₃CH₃ 5.8 1.4 —(CH₂)₅CH₃3.8 2.4

TABLE 22 2.4.5-trichlorophenol ester Carboxylic acid wt. g wt. g

8.3 13.2

0.8 1.2

TABLE 23

2,4,5-Trichlorophenol ester R Wt. (g) Wt. (g) —O(CH₂)₃CH₃ 3.3 4.8—O(CH₂)₄CH₃ 3.0 2.5 —O(CH₂)₅CH₃ 2.3 3.9 —O(CH₂)₂O(CH₂)₃CH₃ 3.3 4.4—O(CH₂)₂OC(CH₃)₃ 1.3 1.9

TABLE 24

2,4,5-Trichlorophenol ester R Wt. (g) Wt. (g) —O(CH₂)₃CH₃ 6.5 5.2—O(CH₂)₂O(CH₂)₃CH₃ 4.9 5.2 —O(CH₂)₂OC(CH₃)₃ 4.6 2.1

TABLE 25

2,4,5-Trichlorophenol ester R Wt. (g) Wt. (g) —O(CH₂)₃CH₃ 2.9 2.5—O(CH₂)₂O(CH₂)₃CH₃ 2.0 1.5 —O(CH₂)₂OC(CH₃)₃ 2.0 1.3

The dideoxy compounds of formula (1) are prepared by removing thebenzylic and aminal hydroxy groups. The process includes subjecting anon-dideoxy compound of formula (1) (wherein R₂ may be hydrogen or acyl)to a strong acid such as trichloroacetic acid, trifluoroacetic acid orborontrifluoride etherate with trifluoroacetic acid being preferred, anda reducing agent, such as sodium cyanoborohydride or triethylsilane,with triethylsilane being preferred. The reaction takes place attemperatures of between −5 and 70° C. and in a suitable solvent such asmethylene chloride, chloroform or acetic acid, with dichloromethanebeing preferred. The acid should be present in an amount of 2 to 60moles per mole of substrate, and the reducing agent should be present inan amount of 2 to 60 moles per mole of substrate. This process affordsselective removal of the aminal and benzylic hydroxy groups.

The compounds represented by the formula (1) have improved propertiesover the previously known N-acyl hexapeptide antifungals. For example,in general the compounds exhibit oral bioavailability, a property whichis important for any systemic antifungal agent. Also, numerous N-acylcompounds of the formula (1) have enhanced antifungal activity andenhanced water solubility.

Among the N-acyl hexapeptides represented by the formula (1) certain arepreferred embodiments of the invention. The compounds wherein R₂ is adiphenyl acyl group

wherein Z is a carbon to carbon bond and R₄ is an alkoxy, cycloalkoxy orcycloalkylalkoxy group are preferred antifungals. Also preferredcompounds are represented when Z is a carbon to carbon bond and R₄ is—Y—R₆ and R₆ is C₁-C₁₂ alkyl phenyl or substituted phenyl and Y is anacetylenic bond.

A further preferred group of N-acyl hexapeptides is represented when Zis a carbon to carbon bond and R₄ is represented by —O—(CH₂)_(p)—W—R₅and wherein W is a piperidine group.

Examples of preferred compounds of the above first mentioned groupinclude 4-(4-alkoxyphenyl)benzoyl wherein the alkoxy group is preferablya C₅-C₁₀ alkoxy group or C₁-C₄ alkoxy substituted by C₃-C₇ alkyl.Examples of such preferred compounds are represented by the formula 1wherein R₂ is 4-(4-n-hexyloxyphenyl)benzoyl,4-(4-n-heptyloxyphenyl)benzoyl, 4-(4-n-octyloxyphenyl)benzoyl,4-[4-(3,3-dimethylbutoxy)phenyl]benzoyl,4-[4-(2-cyclopentyl-ethoxy)phenyl]benzoyl and4-[4-(2-cyclohexyloxyethoxy)-phenyl]benzoyl.

Examples of the second above mentioned preferred compounds wherein R₄ is—Y—R₆ include 4-[4-(phenylethynyl)-phenyl]benzoyl and4-[4-(n-butylethynyl)phenyl]benzoyl.

Examples of preferred compounds of the invention wherein R₄ represents—O—(CH₂)_(p)—W—R₅ are represented when R₂ has the formula

wherein W—R₅ is piperidino, 4-n-propylpiperidino, 4-benzylpiperidino,4-cyclohexylpiperidino, 4-cyclohexylmethylpiperidino, and thepharmaceutically acceptable acid addition salts such as thehydrochloride salts, the sulfate salts or the phosphate salts.

Preferred cyclohexylpeptide compounds are represented by the formula 1wherein R′═R″=methyl, R₁ is hydrogen and R₂ is a preferred acyl group asdefined hereinabove.

Table 26 is a list of the most preferred R₂ substituents, whereinR═R₇═R^(y)═OH; R′═R″═R′″═CH₃; and R₁═H. TABLE 26 Ester A30912A R₂Reactant (g) Nucleus (g) Product (g) FABMS

5.2 6.9 1.4 1142.4951**

2.1 2.5 2.0 1200.5336**

5.2 6.4 1.1 1194.5282* 

2.4 3.3 0.9 1136.4832* 

2.0 3.2 3.0 1194.5213* 

1.3 1.5 2.4 1194.5247* 

4.6 7.4 1.3 1126.5025* 

2.5 3.7 5.1 1140.5103* 

3.5 5.0 1.4 1154.5343* 

4.4 6.7 6.5 1170.5234* 

1.9 2.9 1.4 1170.5261* 

1.8 2.6 0.2 1166.4758* *m + 1;**m + [Li]⁺

The N-acylhexapeptides provided by this invention are useful in thetreatment of fungal infections both systemic infections and skininfections. Accordingly this invention also provides a method fortreating fungal infections in man and animals which comprisesadministering to said host an antifungally effective non-toxic amount ofan N-acyl-cyclohexapeptide represented by the formula 1. A preferredantifungal method comprises administering an N-acylhexapeptide compoundwhere, in formula 1, R′═R″=methyl, R₁ is hydrogen and R₂ is a preferredacyl group as defined hereinabove.

The antifungal compound can be administered parenterally, e.g. i.m.,i.p. or s.c., nasally, orally or can be applied topically for skininfections. The dose administered of course will vary depending on suchfactors as the nature and severity of the infection, the age and generalhealth of the host and the tolerance of a particular host to theparticular antifungal agent. The particular dose regimen likewise mayvary according to such factors and may be given in a single daily doseor in multiple doses during the day. The regimen may last from about 2-3days up to about 2-3 weeks or longer.

This invention also provides pharmaceutical formulations useful foradministering the antifungal compounds of the invention. Theseformulations comprise an N-acylhexapeptide represented by the formula 1or a pharmaceutically acceptable, non-toxic salt thereof and apharmaceutically acceptable carrier.

For parenteral administration the formulation comprises a compound ofthe formula 1 and a physiologically acceptable diluent such as deionizedwater, physiological saline, 5% dextrose and other commonly useddiluents. The formulation may contain a solubilizing agent such as apolyethylene glycol or polypropylene glycol or other known solubilizingagent. Such formulations may be made up in sterile vials containing theantifungal and excipient in a dry powder or lyophilized powder form.Prior to use, the physiologically acceptable diluent is added and thesolution withdrawn via syringe for administration to the patient. Fororal administration, the antifungal compound is filled into gelatincapsules or formed into tablets. Such tablets also contain a bindingagent, a dispersant or other suitable excipients suitable for preparinga proper size tablet for the dosage and particular antifungal compoundof the formula 1. For pediatric or geriatric use the antifungal compoundmay be formulated into a flavored liquid suspension, solution oremulsion. A preferred oral carrier system is lineolic acid, cremophorRH-60 and water and preferably in the amount (by volume) of 8% lineolicacid, 5% cremophor RH-60, and 87% sterile water. The compound is addedto the system in an amount of 2.5 to 40 mg/ml.

For topical use the antifungal compound can be formulated with a drypowder for application to the skin surface or it may be formulated in aliquid formulation comprising a solubilizing aqueous liquid ornon-aqueous liquid, e.g., an alcohol or glycol. Such formulations areuseful forms for use in the antifungal method provided herein.

The N-acylcyclohexapeptides provided herein may be formulated asdescribed above in unit dosage formulations comprising for injectionbetween about 50 mg and about 500 mg per vial. For oral use gelatincapsules or tablets comprising between about 100 mg and about 500 mg percapsule or tablet can be provided.

Preferred formulations of the invention comprises the active ingredientpresented by the formula 1 wherein R′═R″=methyl, R₁ is hydrogen and R₂is 4-[4-(phenylethynyl)-phenyl]benzoyl in gelatin capsules or as activeingredient the antifungal represented by the formula 1 whereinR′═R″=methyl, R₁ is hydrogen and R₂ is4-[4-[2-(4-cyclohexyl-piperidino)ethoxy]phenyl]benzoyl or thehydrochloride salt form thereof in tablet or gelatin capsules. Furtherpreferred formulations are those in which a preferred compound, asdescribed above, is employed.

In yet a further aspect of the present invention there is provided amethod for treating patients suffering from Pneumocystis pneumonia. Themethod can be used prophylactically to prevent the onset of theinfection which is caused by the organism Pneumocystis carinii. TheN-acylcyclicpeptide can be administered parenterally, e.g. viaintramuscular (i.m), intravenous (iv.) or intraperitoneal (i.p.)injection, or orally or by inhalation directly into the airways of thelungs. Preferably the cyclic peptide is administered via inhalation ofan aerosol spray formulation of the compound.

An effective amount of a cyclic peptide will be between about 3 mg/kg ofpatient body weight to about 100 mg/kg. The amount administered may bein a single daily dose or multiple doses e.g. two, three or four timesdaily throughout the treatment regimen. The amount of the individualdoses, the route of delivery, the frequency of dosing and the term oftherapy will vary according to such factors as the intensity and extentof infection, the age and general health of the patient, the response ofthe patient to therapy and how well the patient tolerates the drug. Itis known that PCP infections in AIDS patients are highly refractoryowing to the nature of the infection. For example, in severe, advancedinfections the lumenal surface of the air passages becomes clogged withinfectious matter and extensive parasite development occurs in lungtissue. A patient with an advanced infection will accordingly requirehigher doses for longer periods of time. In contrast, immune deficientpatients who are not severely infected and who are susceptible to PCPcan be treated with lower and less frequent prophylactic doses.

The activity of the cyclicpeptide represented by the formula 1 isdemonstrated in immunosuppressed rats. The tests were carried out ingeneral as follows. One week after initiation of immunosuppression ratswere inoculated intratracheally with parasites and maintained onimmunosuppression for the remainder of the study. Prophylactictreatments began one day after parasite inoculation and therapeutictreatments began 3 or 4 weeks later after moderate PCP developed. Eightor ten animals were assigned to the following groups: those receivingtest compound; non-treated Pneumocystis infected control animals;animals treated with trimethoprim-sulfamethoxazole (TMP-SMX); ornon-treated, non-infected control animals. The efficacy of differenttreatments was evaluated by monitoring animal weights and survivalduring the studies and by determining the severity of PCP at necropsy.Stained impression smears of the lungs and stained lung homogenates wereevaluated to determine the intensity of P. carinii infection.

The immune deficient rats employed in the tests were prepared asfollows. Female Lewis rats weighing from 120-140 g each were immunesuppressed with methyl prednisolone acetate at a dose of 4 mg/100 g forthe first week, 3 mg/100 g for the second week and continuing weeklythereafter at 2 mg/100 g. All rats, except for the non-infected controlrats, were inoculated intratracheally with 0.1 ml to 0.2 ml ofDulbecco's Modified Eagle Media containing between >10⁵ and 10⁶ P.carinii (trophozoites, precysts and cysts) harvested from the lungs ofheavily infected donor animals (infection scores of 6) and maintained ascryopreserved (liquid nitrogen) inocula. Rats were maintained on immunesuppression and PCP was allowed to develop for 3 or 4 weeks beforeinitiation of therapy with test compounds. Body weights were recordedweekly and rats were allocated into treatment groups such that eachgroup had a similar distribution of percent weight loss among animals.Rats were treated with test compounds for 2 or 3 weeks and then werenecropsied. For prophylaxis studies, administration of test compound wasinitiated one day after intratracheal inoculation of parasites and wascontinued until the rats were necropsied.

Following the evaluation period for test compounds, the rats werenecropsied and test results evaluated by Giemsa-stained,silver-methenamine stained impression smears and/or bysilver-methenamine stained lung homogenate (see below). Necropsy wascarried out as follows. The test rats were anesthetized with a mixtureof ketamine hydrochloride and xylazine and then exsanguinated via theright atrium. Internal organs in the abdominal and thoracic cavitieswere examined for gross lesions.

A small portion of lung tissue from the left lobe of each rat was usedto make the impression smears described below. Giemsa-stained impressionsmears were evaluated to determine the total number of parasites(trophozoites, precysts, and cysts). Impression smears from rats ingroups whose treatments exhibited some anti-Pneumocystis activity (asjudged by infection scores from Giemsa-stained slides) and from rats inthe control groups were also stained with methamine silver, a stainspecific for the cyst wall of the organism. Impression smears wererandomized, numbered, and then evaluated. The infection scores used wereas follows: Score Basis 0 No parasites found 1 1 to 5 parasites/10 oilfields 2 ca 1 parasite/field 3 2-10 parasites/field 4 >10 but <100parasites/field 5 >100 but <1,000 parasites/fieldA score of 6 was reserved for those infections with impression smearscontaining >1,000 organisms/field (too numerous to count).Giemsa-stained slides were examined microscopically using a finalmagnification of 1008×. Methenamine silver-stained slides were examinedwith a final magnification of 400×.

Cysts in rat lung tissue were quantified as follows. A small portion oflung tissue from the left lobe of each rat was used to make impressionsmears as described above. The remainder of each lung was weighed,placed in a tube containing Hanks balanced salt solution (HBSS) (40× thelung weight) and homogenized using a Brinkman model tissue homogenizer.Two μ1 samples of the homogenized lung samples (1:4 dilution in HBSS)were placed in wells of teflon-coated, 12-well slides, stained withmethenamine silver, and the number of cysts were scored as describedabove for the impression smears.

The activity and efficacy of two preferred N-acylcyclohexapeptides inthe test animals is presented below. The compound of the formula 1wherein R′═R″=methyl, R₁ is hydrogen and R₂ is4[(4-phenylethynyl)phenyl]benzoyl when administered as an aerosolsolution at a concentration of 5 mg/ml for one hour, twice weekly for 5weeks resulted in 90% reduction in P. carinii cysts in the lungs. Whengiven orally at 10 mg/kg, bid for 3 weeks, the number of cysts in thelungs was reduced by >99% when compared with infected vehicle controls.

When the preferred N-acylcyclicpeptides were administered orally and byintraperitoneal injection the compound was effective in clearing P.carinii cysts from the lungs of heavily infected rats. For example, whenthe compound was administered at 10 or 40 mg/kg, bid for 4, 8 or 12days, the number of identifiable cysts in the lungs of heavily infectedrats was reduced by >99%. Similar efficacy was observed when thecompound was administered i.p. at 1 mg/kg.

When tested orally for prophylactic activity, the preferred compoundexhibited >99% cyst reduction in one of two studies when infectedanimals were dosed at 1 mg/kg and when given higher doses of 5 or 4mg/kg.

Another preferred compound of the invention represented by the formula 1wherein R′═R″=methyl, R₁ is hydrogen and R₂ is4-[4-[2-(4-cyclohexylpiperidino)-ethoxy]phenyl]benzoyl as thehydrochloride salt was also effective in the treatment of PCP. Aerosolprophylaxis (two 60-minute treatments twice a week for 5 weeks), washighly effective. in preventing PCP in the infected immune suppressedrats. Aerosol therapy with 5, 10, 25, or 50 mg/ml of aerosolizedsolution reduced the number of cysts in the lungs by >99% when comparedto controls. Similar results were obtained by i.p. dosage.

The following examples of compounds of the invention and the manner oftheir preparation further describe the present invention.

General Procedure N-Acylation of Cyclohexpeptide Nuclei

The preparation of the derivatives of the A30912A nucleus wasaccomplished by the following general procedure, with Table 27 listingthese derivatives.

The A30912A nucleus, prepared by methods known in the art fromAspergillus rugulosus (NRRL 8113;ATCC 58398) which provides the startingcompound which is then deacylated using Actinoplanes utahensis (U.S.Pat. No. 4,293,482), with 2,4,5-trichlorophenol ester are dissolveddimethylformamide (25-50 ml) and stirred for 17-65 hours at roomtemperature. The solvent is removed in vacuo and the residue is slurriedin ether and collected by filtration. The solid product is washed withmethylene chloride and then dissolved in either methanol oracetonitrile/water (1:1 v/v). This solution is injected on a waters 600Esemi-preparative chromatography system using a Rainin Dynamax-60A C₁₈reverse-phase column. The column is eluted beginning with 20-40% aqueousacetonitrile and 0.5% monobasic ammonium phosphate (w/v) (monitored byUV at 230 rim and at a flow rate of 20 ml/min) until the unreactedA30912A nucleus is eluted and then deleting the buffer and eluting theproduct peak in aqueous acetonitrile. The fraction containing theproduct is evaporated in vacuo or lyophilized to provide the purecompound. The product may be analyzed by the same HPLC instrument usinga Waters C₁₈ Micro Bondapak column and eluting with 40% aqueousacetonitrile containing 0.5% monobasic ammonium phosphate (w/v) at a 2ml/min flow rate and monitoring the UV at 230 nm. The products may alsobe analyzed by fast atom bombardment mass spectrometry (FABMS). (In thecompounds used, R′═R″═R′″=CH₃, R═OH, R^(y)═OH, R₁═H, R₇═OH, and R₂ is asdefined). TABLE 27 Ester A30912A Pro- HPLC Reactant Nucleus ductRetention R₂ (mg) (g) (mg) FABMS (min)

561 1.0 235 1072* 4.08

576 1.0 294 1062* 4.46

579 1.0 355 1086* 5.75

634 1.0 359 1130* 5.79

289 0.5 81 1083* 6.08

594 1.0 295 1098* 6.44

596 1.0 270 1100* 8.15

596 1.0 359 1100* 9.13

596 1.0 301 1100* 10.24

629 1.0 180 1104**

683 1.0 384 1147** 1.92

1490 2.0 116 1195** 2.06

1000 1.2 194 1190*+ 2.41

734 0.9 303 1202* 2.21

810 1.0 230 1187** 2.52

750 1.0 126 1201** 3.50

596 1.0 190 1078** 6.30

571 1.0 295 1058** 7.91

287 0.5 110 1082* 4.52

593 1.0 307 1096* 7.28

313 0.5 104 1324* 19.04

579 1.0 293 1086* 6.14

511 1.0 322 1032* 5.10

514 1.0 287 1034* 6.14

546 1.0 285 1060* 12.48

501 1.0 218 1002** 2.53

291 0.5 98 1088* 3.96

616 1.0 341 1116* 11.56

534 1.0 215 1050*** 7.59

566 1.0 81 1054** 3.89*(m − 1) + [Na]⁺;**(m + 1);***m + [Na]⁺

Compounds such as those listed in Table 27 could be further modified atthe phenolic hydroxy to provide R₇═—OPO₃HNa as shown in Table 28. Theprocedure is as follows:

The lipopeptide (1 equivalent) and tetrabenzylpyrophosphate (2equivalents) were dissolved in dimethylformamide which had been driedover 13× molecular sieves. Lithium hydroxide monohydrate (5 equivalents)was added and the stirred solution was monitored by HPLC. After 0.5 hrand 1 hr more lithium hydroxide (5 equivalents) was added. Between 1 and2 hrs. the reaction was quenched with glacial acetic acid, the solventremoved under vacuum, and the residue purified over a semi-preparativeC18 reverse-phase column using an aqueous acetonitrile eluent. Thepurified product was dissolved in (1/1) acetic acid/water with sodiumacetate (1 equivalent) and 10% Pd/C catalyst. The solution was placedunder an atmosphere of hydrogen gas and stirred for 1 hr. Afterfiltering to remove the catalyst, the solution was lyophilized toprovide the pure final product. The purity was assessed by analyticalHPLC and the product was analyzed by fast atom bombardment massspectrometry (FABMS). TABLE 28 Start. Mat. Prod. R₂ R₇ Wt. (mg) R₇ Wt.(mg) FABMS

—OH 500 —OPO₃HNa 140 1184

—OH 300 —OPO₃HNa 62 1228.4472**m + 1

EXAMPLE 2

(The Scheme following this Example illustrates the process describedherein.)

In the manner described under General Procedure, above, 48.1 g (60.2 mM)of the A30912A nucleus, (A), was mixed with 26.0 g (48.2 mM) of the2,4,5-trichlorophenol ester of[[(4″-pentyloxy)-1,1′:4′,1″-terphenyl]-4-carboxylic acid (B) indimethylformamide (8.5 L) and stirred for 48 hours. Solvent evaporationand purification by HPLC gave 18 g of (C). Analysis by FABMS gave a peakat 1140.5103 (m+1).

In order to protect the aminal hydroxy site, the cyclic peptide is mixedwith 2-(trimethylsilyl)ethanol (100 eq) and p-toluenesulfonic acid (0.1eq) in p-dioxane (0.035 M) and the reaction mixture is stirred atambient temperature for 6-8 hours. After adding 100 mg of solid sodiumbicarbonate, the solvent is removed in vacuo and the residue dissolvedin methanol and passed over a C18 reverse-phase HPLC column to purifythe major component.

In this instance, 2 g (1.75 mM) of compound (C) was mixed with 25 ml(175 mM) of 2-(trimethylsilyl)ethanol and 34 mg (0.175 m) ofp-toluenesulfonic acid with the reaction proceding for 7 hours.Purification by chromatography gives 1.4 g of product (D) which shows asingle peak by analytical HPLC with a retention time of 4.37 min. wheneluting with 70% aqueous acetonitrile at 2 ml/min and monitoring the UVat 280 nm.

The phosphate ester of the protected compound can then be made bytreatment with lithium bis(trimethylsilyl)amide (1.3 eq) in pyridine.After 10 min. tetrabenzylpyrophosphate (1.3 eq) is added and after 15min. the solvent is evaporated. the residue is chromatographed over areverse-phase C18 HPLC column to give the pure product.

In this manner, 1.4 g (1.1 mM) of compound (D) was dissolved in pyridine(18 ml) and 1.4 ml (1.4 mM) of lithium bis(trimethylsilyl)amide (1.0 Min hexane) was added followed in 10 min. by 0.78 g (1.4 mM) oftetrabenzylpyrophosphate. After 15 min. the solvent was evaporated andthe residue was dissolved in methanol and purified over a preparativeHPLC column to give 812 mg of the pure product (E). The material showeda single peak with a retention time of 5.86 min. when analyzed by HPLCusing the previously described system and eluting with 75% aqueousacetonitrile.

The protecting groups can be removed to give the final product by theuse of trimethylsilylbromide (5 eq) in dichloromethane. Water is addeddropwise to the slightly yellow, totally clear solution. As the additionproceeds the yellow color disappears and white precipitate begins toform. When precipitation ceases, the water addition is stopped. Solventis removed in vacuo and the residue is washed with several portions ofdiethyl ether. Water is then added and the mixture is sonicated todissolve the solid. After pentane extraction, the aqueous layer islyophilized to give the product.

Using this, method 722 mg (0.48 mM) of the protected compound (E) wasdissolved in dichloromethane (15 ml). Trimethylsilylbromide (0.32 ml,2.4 mM) was added and, after 15 min, water was added dropwise. Afterprecipitation was complete, the solvent was removed and the residue waswashed with diethyl ether. This solid was dissolved in water and washedwith pentane. Lyophilization of the aqueous layer gave 450 mg ofcompound (F). The product (F) was analyzed by FABMS (using Li+) to givea peak at 1226.4853 (Calculated for C₅₈H₇₄N₇O₂₀PLi=1226.4886). Whenanalyzed by HPLC using a C18 reverse-phase column and eluting with 55%aqueous acetonitrile with 0.5% acetic acid at 2 ml/min and monitoring byUV at 280 nm, the compound had a retention time of 1.72 min.

Preparation of Dideoxy Cyclohexapeptide

The preparation of the dideoxy compounds may be accomplished A thefollowing procedure with Table 29 listing derivatives.

To a suspension of a non-dideoxy cyclohexapeptide (formula (I) whereR═OH and R₂ s hydrogen or acyl), indichloromethane is added the reducingagent triethylsilane in dichloromethane. The solution is stirred and thevolatile components are removed under reduced pressure and the residue,triturated with diethyl ether The compound is purified using HPLC, andthe product lyophilized.

EXAMPLE Dideoxycilofungin

To a suspension of cilofungin (10.00 g, 9.71 mmol) in dichloromethane(100 ml) was added a solution of triethylsilane (96 ml, 602 mmol) indichloromethane (50 ml). Trifluoroacetic acid (46.4 ml, 602 mmol) wasadded as a solution in dichloromethane (50 ml) over 15 minutes. Thesolution was stirred at room temperature for two hours. The volatilereaction components were removed under reduced pressure and the residuetriturated with diethyl ether. The compound was purified by reversedphase HPLC by means of a “Prep LC/System 500” unit (Waters Associates,Inc., Milford, Mass.) using a Prep Pak 500/C₁₈ Column (WatersAssociates, Inc.) as the stationary phase. The column eluted with agradient mobile phase using CH₃CN/H₂O (10:90 to 20:80 v/v) at 500 psi.The product containing fractions were pooled, evaporated under reducedpressure, and lyophilized from p-dioxane to yield dideoxycilofungin(6.66 g, 68.7%). FAB-MS: m/z calc. for C₄₉H₇₂N₇O₁₅, 998.5086; found,998.512; UVλ (EtOH)nm(ε) 202.60(61012), 256.20(18569).

Table 29, indicates R₂, the amount of the cyclic hexapeptide andreagents, and yield of dideoxy compounds prepared as described above.(R′═R″═R′″CH₃, R₁═H and R═R^(y)═R₇═OH); T.E.S.=triethylsilane;.TFA=trifluoroacetic acid; numbers are weights in grams). TABLE 29Starting R₂ Material TES TFA Yield

0.500 0.256 0.251 0.095

0.500 2.47 2.42 0.063

0.500 2.63 2.57 0.392

2.00 9.49 9.72 1.47

0.500 3.50 3.44 0.291

1-43. (canceled)
 44. A pharmaceutical formulation comprising a compoundhaving the following formula:

wherein R is —O(CH₂)₃CH₃, —O(CH₂)₄CH₃, —O(CH₂)₅CH₃, —O(CH₂)₂O(CH₂)₃CH₃,or —O(CH₂)₂OC(CH₃)₃, and a pharmaceutically acceptable carrier, whereinthe formulation is formulated for inhalation:
 45. The pharmaceuticalformulation of claim 44, wherein the formulation comprises an aerosolspray.
 46. A method for inhibiting parasitic activity comprisingcontacting a formulation of claim 44 with a parasite via inhalationadministration.
 47. A method of inhibiting fungal activity comprisingcontacting a formulation of claim 44 with a fungus via inhalationadministration.
 48. A method of inhibiting the growth of organismsresponsible for opportunistic infections in immunosuppressed individualscomprising administering a formulation of claim 44 to said individualvia inhalation.
 49. A method of inhibiting the growth of Pneumocystiscarinii comprising contacting a formulation of claim 44 withPneumocystis carinii via inhalation.
 50. The method of claim 49, whereinthe formulation comprises an aerosol spray.
 51. The method of claim 49,wherein administration of the formulation is prophylactic.
 52. Themethod of claim 50, wherein administration of the formulation isprophylactic.